|Year : 2017 | Volume
| Issue : 2 | Page : 56-67
Antifibrotic effects of Punica granatum peels through stimulation of hepatic stellate cell apoptosis in thioacetamide-induced liver fibrosis in rats
Abdel Razik H Farrag1, Enayat A Omara1, Asmaa F Galal2, Sayed A El-Toumy3, Nabila S Hassan1, Hafiza A Sharaf1, Somaia A Nada4
1 Department of Pathology, National Research Centre, Cairo, Egypt
2 Department of Toxicology and Narcotics, National Research Centre, Cairo, Egypt
3 Department of Chemistry of Tannins, National Research Centre, Cairo, Egypt
4 Department of Pharmacology, National Research Centre, Cairo, Egypt
|Date of Submission||12-Jun-2017|
|Date of Acceptance||17-Sep-2017|
|Date of Web Publication||29-Dec-2017|
Abdel Razik H Farrag
Pathology Department, Medical Research Division, National Research Centre, 33 El-Buhouth Street, 12622 Dokki, Cairo
Source of Support: None, Conflict of Interest: None
Background/aim Liver fibrosis is a major global health problem. The present study aimed to evaluate the antioxidant and antifibrogenic potential of Punica granatum peels extract against thioacetamide (TAA)-induced hepatic fibrosis.
Materials and methods Rats were divided into six groups. Group 1 was the control; group 2 was injected with TAA (150 mg/kg, intraperitoneal) (fibrosis group) for 4 weeks; group 3 received P. granatum peels extract only (200 mg/kg); group 4 rats were given oral sliymarin (50 mg/kg) for 4 weeks after withdrawal of TAA; groups 5 and 6 rats were given oral P. granatum peels extract (100 and 200 mg/kg) for 4 weeks after withdrawal of TAA. Fibrosis was assessed histologically and by measuring the hepatic hydroxyproline content. The degree of liver fibrosis was assessed by Masson’s trichrome staining and α-smooth muscle actin as the marker of the activated hepatic stellate cells was detected immunohistochemically. Serum markers of liver damage and oxidative stress were also assessed.
Results The biochemical analyses have shown that P. granatum peels extract or sliymarin significantly reduced the progression of hepatic fibrosis. The plant extract or sliymarin resulted in a significant improvement of liver damage by the reduced levels of serum alanine aminotransferase and alkaline phosphatase. Oral administration of P. granatum peels or sliymarin has also restored normal levels of malondialdehyde, hydroxyproline content as markers of fibrosis content (P<0.05) in the liver, and retained control activities of endogenous antioxidants such as superoxide dismutase, nitric oxide, and glutathione. The histological evaluation showed that the plant extract or silymarin treatment maintained the architecture of the liver nearly normal and attenuate the accumulation of excessive collagen in the liver fibrosis caused by TAA. We also observed that P. granatum peels extract or silymarin-treated rats reduced α-smooth muscle actin.
Conclusion The obtained results have shown that P. granatum peels extract effectively blocked hepatic stellate cell proliferation and they may be beneficial in the treatment of liver fibrosis.
Keywords: fibrosis, histopatholgy, immunohistochemistry, Punica granatum, thioacetamide
|How to cite this article:|
Farrag AH, Omara EA, Galal AF, El-Toumy SA, Hassan NS, Sharaf HA, Nada SA. Antifibrotic effects of Punica granatum peels through stimulation of hepatic stellate cell apoptosis in thioacetamide-induced liver fibrosis in rats. J Arab Soc Med Res 2017;12:56-67
|How to cite this URL:|
Farrag AH, Omara EA, Galal AF, El-Toumy SA, Hassan NS, Sharaf HA, Nada SA. Antifibrotic effects of Punica granatum peels through stimulation of hepatic stellate cell apoptosis in thioacetamide-induced liver fibrosis in rats. J Arab Soc Med Res [serial online] 2017 [cited 2018 Feb 25];12:56-67. Available from: http://www.new.asmr.eg.net/text.asp?2017/12/2/56/221879
| Introduction|| |
Liver fibrosis is a major global health problem causing ∼1.4 million deaths per year . It is the common pathologic result of all chronic liver diseases. Its main causative factors in developing countries are diseases with hepatitis B and C viruses and parasitic infection with Schistosoma mansoni, while it can be excessive alcohol consumption in developed countries . In addition, hepatotoxic drugs [antibiotics, carbon tetrachloride, thioacetamide (TAA), and acetaminophen] consequently result in liver fibrosis . The beginning of the hepatotoxic effect of TAA demands metabolic activation and cases of liver fibrosis ,,. For the study mechanism of hepatic fibrogenesis and potential antifibrosis we used TAA as the model for liver fibrosis. The injection TAA in animals gives rise to centrilobular necrosis, apoptosis, and periportal inflammatory cell infiltration and fibrosis in the liver . The development of liver fibrosis is clearly produced with abnormal liver architecture resulting in intense changes of intrahepatic/extrahepatic hemodynamic and finally impairment of the liver function . Liver fibrosis is the result of progressive extracellular matrix (ECM) accumulation, distinguished by scar tissue replacement and regenerative nodules occurring in the hepatic perisinusoidal space .
Studies over earlier period have focused on the mechanism of fibrosis and fibrogenic cells that create the scarring reaction known as hepatic stellate cells (HSCs). The activation of HSCs and transformation to myofibroblast-like cells then proliferate and produce an ECM with continual chronic inflammation . Oxidative stress, the result of the imbalance between production and clearance of reactive oxidative species (ROS), appears to be a common feature in the different types of liver injuries . In-vitro and in-vivo data have suggested the participation of ROS in the pathogenesis of fibrosis ,. ROS with inflammatory cytokines and growth factors induce HSC activation. Therefore, any interference aimed at reducing the exposure of HSCs to these oxidative and inflammatory stimuli could slow down or inhibit the progression of fibrosis induced by inflammatory cytokines or mediators; activated stellate cells advance hepatic fibrosis and consequently disturb the circulating blood flow in the liver. Physiologically, these pericytes secrete α-smooth muscle actin (α-SMA) that is responsible for connective tissue formation in response to liver injury, and α-SMA is commonly used as a marker of myofibroblast formation ,. Liver lesions from fibrosis are hard to cure, but clinical interventions may block further development or reduce the associated complications . However, current medications for managing hepatofibrosis are limited due to their undesirable side effects . Liver fibrosis could be considered a bidirectional process and could be reversible . The hope is that if antifibrotic therapy can reconstitute the normal balance of the liver, normal function can be restored and clinical appearance may retract. Current and developed approaches primarily target to inhibit the activated HSCs, proliferation, and products as well as to enhance their apoptosis .
Medicinal plants have been used from ancient times for the treatment of a large variety of diseases  as well as for hepatotoxicity . In fact, this is owing to its lower costs and greater compatibility  and being rich in various compounds such as triglycerides, flavonoids, and polyphenols that can protect the liver against damages induced by hepatotoxic drugs . Foods rich in natural antioxidants have been choose as an agent to prevent and cure liver damage . Pomegranate peel is known for its abundant health-promoting qualities and apparent wound-healing properties , anticancer property , antiatherosclerotic, antioxidative capacities , antiviral , antifungal , and antibacterial benefits . This is due to the fact that pomegranate contains large amounts of polyphenols and flavonoids . Furthermore, recent numerous studies have proved the hepatoprotective property of pomegranate that possesses definite hepatoprotective properties, making it a significant therapeutic agent in the treatment of hepatic fibrosis and oxidative damage . The present study was designed to evaluate the antifibrogenic effect of P. granatum peels extract on liver fibrosis induced by TAA in rats along with the observation of any potential changes in the biochemical marker, HP as fibrosis markers, histopathological features, and expression of the activated HSCs (SMA).
| Materials and methods|| |
Preparation of the P. granatum extract
Fresh P. granatum L. fruits were collected from Upper Egypt (October 2015). The peels were separated manually from the fruit and then washed with water, cut into small pieces, and sun dried until complete dehydration. Dried peels were ground into a fine powder in a mortar. The dry powder (50 g) was extracted with 300 ml aqueous 70% methanol in a Soxhlet apparatus for 72 h. The extract was filtered and concentrated to dryness under reduced pressure in a rotary evaporator at 40–50°C yielding 14.5% (w/w) plant extract. The obtained P. granatum L. peels alcoholic extract was stored at 5°C until usage. The plant extract was suspended in warm, distilled water (100 mg/1 ml) and was given orally through a stomach tube to rats at a dose of 100 and 200 mg/kg .
Preparation of drugs
TAA was purchased from Sigma (St Louis, Missouri, USA). Other chemicals and reagents were of high analytical grade and were purchased from standard commercial suppliers. TAA was prepared freshly by dissolving it in sterile distilled water and stirred well until all crystals were dissolved.
Experimental animal procedures
All experimental procedures were performed according to the institutional committee of the animal’s care and use guidelines, National Research Centre (Egypt). Male Sprague-Dawley rats (130–150 g body weight) was obtained from the Animal House of the National Research Centre (Egypt). They were maintained under controlled conditions of temperature 37±5°C and kept at 12 h natural day light and dark night cycles. They were provided standard rats feed and water ad libitum.
Thirty-six rats were randomly divided into six groups (six rats of each) as follows:
- Group 1: served as normal control and received sterile, distilled water only.
- Group 2: TAA group in which rats were intraperitoneally injected with TAA (150 mg/kg) for 4 weeks to induce liver fibrosis in rats.
- Group 3: rats were orally given P. granatum peels (200 mg/kg).
- Group 4: sliymarin group and given oral dose (50 mg/kg) for 4 weeks after withdrawal of TAA.
- Groups 5 and 6: rats were given oral P. granatum peels extract (100 and 200 mg/kg, respectively) for 4 weeks after withdrawal of TAA.
After 24 h of the last injection, blood samples were collected from the retro-orbital plexus after light anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneal). Serum was separated by centrifugation at 3000g for 10 min and was used for the assessment of liver functions. Rats were sacrificed by cervical dislocation, and livers were removed. A portion of liver tissue was washed and homogenized to obtain a 20% (w/v) homogenate, which was used for the assessment of oxidative stress and fibrogenic markers. Another portion was placed in formalin for histopathological and immunohistochemical examinations.
Serum concentrations of alanine aminotransferase (ALT) and alkaline phosphatase activity (ALP) were determined according to the methods of Reitman and Frankel  and Belfield and Goldberg , respectively, using available commercial kits of Biodiagnostics Co. (Cairo, Egypt).
Hepatic oxidative stress markers
The supernatant obtained by centrifugation of the 20% homogenate was used for the assessment of oxidative stress markers. Lipid peroxidation was determined by estimating the level of thiobarbituric acid reactive substances measured as malondialdehyde (MDA), according to the method of Mihara and Uchiyama . Reduced glutathione (GSH) content was determined according to the method of Beutler et al.  and expressed as mg/g wet tissue. Tissue nitric oxide (NO) metabolites were determined according to the method described by Miranda et al.  and expressed as µmol/l/g wet tissue. In addition, superoxide dismutase (SOD) activity was determined by a kinetic method using a commercial kit of Ransel, Randox Laboratories Co. (Antrim, UK).
Marker of hepatic fibrosis
Hepatic collagen content was assessed biochemically through the determination of hydroxyproline (HP) concentration, according to the method of Woessner . Results were expressed as µg/g wet tissue.
After fixation of liver tissues obtained from rats in the studied groups in 10% formal saline for 24 h, they washed in tap water. Then, serial dilutions of alcohol were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56° in oven for 24 h. Paraffin wax tissue blocks were prepared for sectioning at 4 μm thickness by rotary microtome. The obtained tissue sections were collected on glass slides, deparaffinized, and were stained by hematoxylin and eosin stains. After that, examination was done using light electron microscope .
Masson trichrome stain for collagen fibers
Masson trichrome stain was used for demonstrating the collagen fibers .
Immunostaining for α-SMA was performed on paraffin sections from the livers of all groups. This was done using a primary antiserum to α-SMA (1 : 100) followed by biotinylated horse antimouse antiserum, avidin–biotin complex, and DAB as the chromogen. Smooth muscle was used as positive control specimens. On the other hand, one of the liver specimens was used as negative control by omitting the step of applying the primary antibody. A positive reaction was expressed as a dark brown color in the cytoplasm of hepatocyte stellate cells indicating its activation into myofibroblasts.
The morphometric measurements were done using Leica Quin 500 Image Analyzer (Leica Imaging systems Ltd, Cambridge, UK) in the Pathology Department, National Research Centre, Cairo. The morphometric measurements were carried out with optical magnification of ×20 on hematoxlin and eosin and Masson trichrome stained sections for the measurement of damaged and fibrotic areas. Ten fields were selected randomly for measurements and the results were expressed in μm2 and SE.
The percent protection with each extract dose was also calculated by the following formula :
where DA is the damaged area.
The results are expressed as mean±SE. Multiple comparisons were performed using one-way analysis of variance followed by Tukey–Kramer as a post-hoc test. A P value of less than 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism software (version 6) (GraphPad Software, Inc., La Jolla, CA, USA).
| Results|| |
P. granatum peels extract treatment attenuated thioacetamide-induced hepatic damage
As compared with the control group, serum ALT levels and ALP activity were significantly elevated in the TAA group. Notably, serum ALT levels and ALP activity were nearly normalized in the P. granatum peels extract cotreated group in a dose-dependent manner. Similarly, rats cotreated with silymarin showed nearly normal levels of ALT and ALP activity as compared with the TAA group ([Table 1]).
|Table 1 Alanine aminotransferase and alkaline phosphatase levels in rats treated with thioacetamide and P. granatum extract|
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P. granatum peels extract treatment attenuated thioacetamide-induced oxidative stress
Hepatic GSH levels and SOD activity were markedly reduced while the liver content of MDA was significantly elevated in the TAA group compared with the control group. Interestingly, P. granatum peels extract or silymarin cotreatment returned both GSH and MDA levels to the nearly normal levels and restored SOD activity, thus protecting against TAA-induced oxidative liver damage. Hepatic levels of NO were markedly increased in rats treated with TAA. Coadministration of P. granatum peels extract or silymarin decreased NO levels, dose-dependently, to nearly normal levels as compared with the control group ([Table 2]).
|Table 2 Glutathione, superoxide dismutase, malondialdehyde, nitric oxide. and hydroxyproline activities in rats treated with thioacetamide and P. granatum extract|
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P. granatum peels extract treatment attenuated thioacetamide-induced hepatic fibrogenesis
Moreover, the antifibrotic effect of P. granatum peels extract or silymarin was further confirmed by biochemical determination of HP levels. As seen from [Table 2], the increase in HP levels induced by TAA was markedly reduced by P. granatum peels extract or silymarin cotreatment in a dose-dependent way. These results confirmed those obtained from histological examination.
Histology of the liver sections of control rats showed normal hepatic cells with well-preserved cytoplasm, central veins, prominent nucleus, and blood sinusoids ([Figure 1]a). Histopathological examination of the livers of TAA group (the fibrosis group) showed disorganization of the normal lobular pattern. Variable degree of degeneration and necrosis were demonstrated starting around the central veins and then progressed to all zones of the hepatic lobules (centrilobular). The affected hepatocytes were vacuolated or fatty degeneration changes, apoptotic having darkly eosinophilic cytoplasm with pyknotic nuclei. Mononuclear cellular infiltration was found between the hepatocytes and marked within the portal areas. Moreover, thick bundles of collagen fibers were bridging the expanded portal areas and central vein and surrounding the lobules ([Figure 1]b). P. granatum peels extract group (200 mg/kg) revealed no histopathological changes and the structure was nearly normal ([Figure 1]c). In the group treated with TAA and silymarin dose (50 mg/kg), the liver tissue exhibited apparent nearly normal hepatic parenchyma with only a few tiny bundles of collagen fibers and with inflammatory cell infiltration ([Figure 1]d). The TAA and P. granatum peels extract (100 mg/kg) group showed mild necrosis around the central vein, moderate fatty degeneration of the hepatocytes, and the collagen fibers were thinner than those noticed in the TAA group ([Figure 1]e). The P. granatum peels extract (200 mg/kg) treated groups showed markedly reduced deposition of fibrous tissue in the liver and tissue morphology nearly similar to the control group ([Figure 1]f).
|Figure 1 Micrograph of liver sections stained with hematoxylin and eosin (H&E): (a) the control group showing normal hepatic cells with well-preserved cytoplasm, central veins (CV), prominent nucleus (N) and visible sinusoids (S), (b) thioacetamide (TAA) group showing distortion of the hepatic architecture, extensive intralobular fibrosis of both porto-portal and porto-central bridging fibrosis with collagen septa formation, massive number of inflammatory cells infiltration (arrow), necrotic hepatocytes (arrowhead) with severe ballooning degeneration (star), (c) P. granatum peels extract (200 mg/kg) showing normal hepatic architecture and preserved lobular pattern with the CV and rounded nuclei (N), (d) TAA and silymarin showing maintained hepatic architecture with minimal damage, mild necrosis around central vein (arrowhead), mild vacuolar degeneration of hepatocytes (arrowhead). and thin collagenous septa formation with inflammation (arrow), (e) TAA and P. granatum peels extract (100 mg/kg) showing partially preserved hepatocytes, moderate necrosis around the central vein (arrowhead), mild vacuolar degeneration of hepatocytes (H), activation of Kuppfer cell (K) and the collagen fibers were thinner than those noticed in the TAA group (arrow), and (f) TAA and P. granatum peels extract (200 mg/kg) showing minimal damage in the hepatic lobule. The necrotic areas of hepatocytes were replaced by normal cells with thin collagenous septa formation (arrow) (H&E, ×400).|
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Masson’s trichrome staining
Masson trichrome stain is an important marker of liver fibrosis and is used for demonstrating collagen fibers. The connective tissue was demonstrated as a thin layer of collagen fibers in the wall of the central vein and the portal tracts ([Figure 2]a). The TAA group displays intense grade of collagen fiber bridge and a pseudolobule structure ([Figure 2]b). The P. granatum peels extract (200 mg/kg) showed few collagen fibers ([Figure 2]c). The TAA group and silymarin (50 mg/kg) showed mild collagen fibers ([Figure 2]d). The group that received P. granatum peels extract (100 mg/kg) demonstrated less collagen fibers than that observed in the TAA-intoxicated rat, but was not of normal level ([Figure 2]e). By coadministration of P. granatum extract (200 mg/kg) the collagen fibers in the treatment groups were markedly reduced in the degree of liver fibrosis ([Figure 2]f).
|Figure 2 Micrograph of liver sections stained with Masson’s trichrome. Collagen can be visualized by the blue color in stains: (a) the control group showing normal degree of collagen fibers in the central vein and portal tract area; (b) thioacetamide (TAA) group showing extensive intralobular collagen deposition of both porto-portal and porto-central bridging fibrosis apparently as intense blue-stained collagen fibers content in the tissue, (c) P. granatum treated group showing nearly normal blue-stained collagen fibers content in the tissue, (d) TAA and silymarin liver shows minimal degree of collagen fibers, (e) TAA and P. granatum (100 mg/kg) group showing moderate collagen fibers surrounding the central vein, (f) TAA and P. granatum (200 mg/kg) group showing minimal collagen fibers surrounding the central vein (Masson’s trichrome, ×400).|
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The mean±SE damaged areas of TAA, TAA and silymarine, TAA and P. granatum peels extract (100 mg/kg), and TAA and P. granatum peels extract (200 mg/kg) are 2561.58±702.44, 779.65±140.17, 667.75±51.98, and 479.72±87.42, respectively ([Figure 3]). The data showed significant decrease in the damaged areas of TAA and silymarin, P. granatum peels extract (100 mg/kg), and TAA and P. granatum peels extract (200 mg/kg) as compared with the TAA group. The percentages of reduction are 69.56, 73.93 and 81.27%, respectively ([Figure 3]).
|Figure 3 Damaged area (μm2) of liver of different groups. Data presented as mean±SE. *Significant decrease as compared with TAA group.|
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The mean±SE fibrotic areas of control, plant, TAA, TAA and silymarine, TAA and P. granatum peels extract (100 mg/kg), and TAA and P. granatum peels extract (200 mg/kg) are 71.92±10.53, 81.83±11.35, 2247.22±263.66, 1931.57±193.24, 1052.12±133.52, and 784.38±63.68, respectively ([Figure 4]). The data showed significant increase in the fibrotic areas of TAA as compared with the control group. The percentage of elevation is 92.34% and significant decrease in the fibrotic areas of TAA and silymarin, TAA and P. granatum peels extract (100 mg/kg), and TAA and P. granatum peels extract (200 mg/kg) as compared with the TAA group. The percentages of reduction are 14.04, 53.18 and 65.50%, respectively.
|Figure 4 Fibrotic area (μm2) of different groups. Data presented as mean±SE. *Significant increase as compared with the control group. **Significant decrease as compared with the TAA group.|
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Immunohistochemical evaluation of α-smooth muscle actin expression
In liver diseases, the major source of matrix proteins produced is to activate HSCs. Therefore, we evaluated immunohistochemical staining of α-SMA as a marker of HSC activation. α-SMA immunoexpression on HSCs were expressed in the form of dark and light stained brown areas in their cytoplasm and was considered a marker of their activation to myofibroblasts. The control group and P. granatum extract showed negative expression of α-SMA staining ([Figure 5]a and [Figure 5]c). The TAA-intoxicated rat demonstrated intense reaction of α-SMA immunostaining in the central and portal tract area. The cytoplasm was stained dark brown color ([Figure 5]b). α-SMA expression in the TAA+P. granatum peels extract (100 and 200 mg/kg) ([Figure 5]e and [Figure 5]f) or silymarin-treated groups showed a reduction compared with TAA in a dose dependent manner indicating inhibition of HSC activation ([Figure 5]d).
|Figure 5 Micrograph of liver sections staining with immunohistochemistry for α-smooth muscle actin (α-SMA) can be visualized by the brown color: (a) the control group showing a limited to the wall of the central vein expression of α-SMA; (b) thioacetamide (TAA) group showing intense immunopositive reaction of α-SMA in a fibrous band connecting between portal and central areas as brown color; (c) P. granatum group showing normal expression of α-SMA positive staining; (d) TAA and slymarin showing reduction in the areas of α-SMA reaction around the central vein; (e) TAA and P. granatum (100 mg/kg) showing moderate α-SMA reaction around the central vein; and (f) TAA and P. granatum (200 mg/kg) showing minimal α-SMA reaction around the central vein (α-smooth muscle actin, ×400).|
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| Discussion|| |
Hepatic fibrosis is known as a massive pathological process that includes various cellular and molecular proceedings that lead to deposition of excess matrix proteins in the extracellular space including collagen . The propose of several antifibrotic treatment effect is particularly studied to downregulate hepatic inflammation, HSC activation, and to increase matrix degradation .
The importance of medicinal plants come from a significant achievement therapy of liver fibrosis ,. Traditional plant drugs have been established to be effective in preventing fibrogenesis and other chronic liver injuries which have potential for controlling liver fibrosis, cirrhosis, and hepatocarcinogenesis ,. Flavonoids have beneficial effects as their strong antioxidant characteristic can preserve the body from free radicals and oxidative stress . Besides, many researchers reported using natural antioxidants, such as vitamin E, resveratrol, quercetin and N-acetyl cysteine and flavonoids suppression of stellate cell activation . Therefore, flavonoids are convenient to display an essential fundamental biological role, especially due to their capability to scavenge ROS .
In the present study, we focused on the antifibrotic effects of P. granatum peels by measuring HP; Masson’s trichrome staining for collagen content, and immunohistochemical detection of α-SMA as the marker of the activated HSCs. Moreover, the present study was to assess whether dietary P. granatum peels extract reduce the progress of liver fibrosis in TAA-intoxicated rats.
The stimulus of fibrosis by TAA occurs in vivo and is consider including the producer of oxidative stress , with the imbalance between fibrosis and antifibrosis signaling pathways. In the current work, the TAA group revealed hepatic damage manifested by significant increase (P<0.05) in serum liver biomarkers (ALT and ALP) when compared with normal control rats. The elevation serum levels of AST and ALT are due to the injury to the impartiality of the liver tissue, since these enzymes normally exist in the cytoplasm and are released to the blood circulation following cellular damage . Hajovsky et al.  have reported that free radicals produced by TAA affect the cellular permeability of hepatocytes leading to increased levels of serum biochemical parameters such as ALT and AST. The treatment with P. granatum peel extract or silymarin effectively ameliorated the significant elevation in serum ALT and ALP activities in TAA administered rats. These findings are in agreement with those of Abdel Rahman et al.  who found that the pomegranate peel extract significantly decreased the damaging influence of CCl4 on the liver. Silymarin showed a marked protection of serum AST and ALP levels in CCl4 induced hepatotoxicity due to the high content of flavonoids ,. It was indicated that hepatotoxins with TAA resulted in liver injury by producing free radicals, which then interact with cellular lipids to enhance lipid peroxidation . The elevated MDA level in TAA rats resulted in the current work also agree with this result; however, treatment with P. granatum markedly reduced the level of MDA as compared with the TAA group. These effects were approved by Fadhel and Amran  and Ajaikumar et al.  who show that the tissue lipid peroxidation level was reduced in the P. granatum extract treated groups of animals as compared with the TAA group. Besides, silymarin shows noticeable preservation of the liver from raising MDA after being toxicated with CCl4 .
Reduced glutathione is a great endogenous antioxidant system that is located in specially high concentration in the liver, and it is known to have key functions in protective processes. The decreased form of GSH becomes easily oxidized to glutathione disulfide on interaction with free radicals. Progressive induced of free radicals produces the oxidative stress, which leads to damage of macromolecules, for example, lipids and can increase lipid peroxidation in vivo .
Intoxicated rats with TAA exhibited a significant decrease in the activity of the antioxidant enzymes SOD and GSH concentration as compared with normal control. These are in agreement with Cruz et al.  who reported that TAA significantly decreased the activity of the antioxidant enzymes (GST, CAT, and SOD) and GSH concentration Uskokovic-Markovic et al.  have recorded that TAA resulted in the elevation of oxidative stress, enhancing free radical-mediated damage to proteins, lipids, and DNA. Treatment with P. granatum or silymarin exhibited significant increase in the GSH and SOD contents when compared with TAA-induced fibrosis. P. granatum and guava leaves have potential antioxidant activity which may be caused due to a lot of phenolic compounds and high antioxidant activity ,.
The phytochemical study of pomegranate peel showed the presence of flavonoids, steroids, terpenoids, and tannins ,. The flavonoids have the ability to decrease xenobiotic that produces hepatic damage in animals and oppose the damaging effects of oxidative stress, collaborating with natural systems such as glutathione and other endogenous protective enzymes .
In the present study, treatment of TAA group with silymarin showed a significant increase in the activity of the antioxidant enzymes SOD and GSH levels. These results are in agreement with Amin et al.  and Padhy et al.  who stated that the treatment of TAA-intoxicated rats with silymarin or/and Calotropis procera caused a significant increase in the activity of the antioxidant enzymes. Moreover, silymarin has powerful antioxidant properties because it acts as a scavenger of the free radicals that induce lipid peroxidation, and has great effect on enzyme systems associated with glutathione and superoxide dismutase .
The NO radicals play a marked role in producing inflammatory response and their toxicity multiplies only when they interact with O2• − radicals to form peroxynitrite that causes damages to biomolecules such as proteins, lipids, and nucleic acids ,. In the present results, P. granatum extract restores the normal level of NO that increased in the TAA group; however, this result is convenient with the result obtained with the study sated that the pomegranate juice was active and it might have an extremely powerful and novel therapeutic effect for scavenging of NO and may also extend their effects on the regulation of pathological conditions caused by progressive generation of NO and its oxidation product peroxynitrite .
Histologically, in our study, TAA injected rats produced severe centrilobular necrosis; hepatocyte vacoulation also scattered inflammatory cell infiltration and fibrosis. These results came in agreement with Ahmed et al. , who reported that the liver sections of TAA-treated animals exhibit hepatic cells with intense toxicity distinguished by centrilobular necrosis, preiportal hepatocyte vacoulation with clearing of cytoplasm, dispersed inflammation, and cell diversion. In addition, these results were nearly similar with numerous past studies, which examine the production of liver fibrosis and cirrhosis by TAA in experimental animals ,. However, in experimental studies, liver fibrosis is commonly induced with TAA, which is readily metabolized to reactive acetamide and TAA-S-oxide. The metabolites formed combine covalently with macromolecules of the hepatic tissue leading to accumulation of fatty acids, damage of proteins and DNA, and formation of ROS. All these compounds impair the endogenous antioxidative system in the liver and are responsible for persistent oxidative stress ,.
All these histopathological changes have improvement after treatment with P. granatum peel extract or silymarin as compared with the TAA group. The use of P. granatum peels extracts reduced the BDL-induced liver fibrosis and improves the liver structure and function . Moreover, these findings are in agreement with Abdel Rahman et al. , Sadia et al. , and Ibrahim  who found that administration of pomegranate peel extract significantly reduced the liver damage and able to improve hepatic steatosis induced by CCl4. The pomegranate peels extract significantly suppress ferric nitrilotriacetate produced oxidative stress and also prevent necrosis and other pathological changes and preserve the hepatic architecture . Similar hepatoprotective effects have been reported with pomegranate peels extract, which inhibited CCl4, or pentachlorophenol-induced oxidative stress and hepatic injury ,.
Pomegranate juice and peel, seeds extracts have antioxidant potential due to high the content of polyphenolic compounds  and possess a potent antioxidant activity , and due to their active compounds that have certain electron donors, which can react with the free radicals to change them to more stable products and terminate the radical chain reaction ,,. In addition, pomegranate peels fundamentally have phenolics, inclusive mainly of hydrolysable tannins (ellagitannins), such as oligomers and punicalagin/punicalin . The treatment with silymarin exhibited improvement of the liver tissue and absence of centrilobular or bridging necrosis .
In the present study, increases of collagen content in TAA-treated animals were observed in all liver tissues. HSC, which is the central mediator in the pathogenesis of fibrosis, are known to be activated by free radicals and increase the ECM with collagen . Mean area percent of collagen fibers was used as an index for assessing the extent of liver fibrosis . However, P. granatum peel extracts markedly decreased the collagen fiber content as compared with the fibrosis group induced by TAA. Moreover, there was statistically significant difference between the TAA group (fibrosis group) and the treatment group (P. granatum peels 100 and 200 mg/kg) based on quantitative morphometric analysis results.
The treatment of mice with CCl4 and pomegranate peel caused a detectable decrease of the collagen fibers; these effects could be related to its antioxidant, antifibrotic, and antiapoptotic properties . Various studies proved that oxidative stress plays a vital role in liver fibrosis . Antioxidants are effective for preventing liver fibrogenesis . Moreover, pomegranate peel antioxidant and antifibrotic properties may be having powerful therapeutic value in preserving liver tissues from fibrosis and oxidative injury . Therefore, P. granatum peels treatment may be helpful as therapy with consideration to antihepatofibrotic properties.
It is commonly known that HSC activation plays a pivotal role in the process of hepatic fibrogenesis, and α-SMA is a marker of activated HSCs . In the present study, immunohistochemical observations of α-SMA was increased in rats injected with TAA and after treated with P. granatum peel extract or silymarin noticeably suppressed α-SMA; however, this indicated suppression of the activation of HSCs.
The antioxidant influence of flavonoids in pomegranate peels extract increases the process of regeneration. This might be due to destruction of free radicals, supplying a competitive substrate for unsaturated lipids in the membrane and/or progress the restoration mechanism of damaged cell membrane . Additionally, flavonoid compounds have a stronger antioxidative activity and can exert their protective role by regulating cell apoptosis; these compounds can inhibit normal hepatic cell apoptosis while accelerating apoptosis of tumor cells and necrotic hepatic cells. Flavonoid compounds also protect liver against damage via modulation of cell mitosis and proliferation as well as the secretion of enzymes against platelets during coagulation and inflammation ,,.
| Conclusion|| |
We have confirmed that P. granatum peels extract has obvious hepatoprotective and antifibrotic powerful effects against TAA-induced fibrosis using an in-vivo model. This helped to clarify the potential antioxidant efficacy of P. granatum peels extract and its ability to suppress HP, collagen content as well as α-SMA. Furthermore, liver function including that of ALT, ALP, and antioxidant enzymes (GSH, SOD, and NO) were improved after P. granatum peels extract treatment. Besides, the obtained results showed that P. granatum peels extract effectively blocked HSC proliferation and they may be beneficial in therapeutic liver fibrosis. In addition, we demonstrated the potent antifibrotic role of flavonoid compounds existent in P. granatum peels extract providing a theoretical basis for its clinical application and indicating an alternative method for the clinical treatment of liver fibrosis.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]